UnGE promoter sequence and its uses

ABSTRACT

The present invention relates to the field of biotechnologies and in particular to the use of a nucleotide sequence having transcription promoter activity in prokaryotic and eukaryotic cell systems.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 16/715,451filed on Dec. 16, 2019, the content of which is incorporated herein byreference in its entirety.

STATE OF THE ART

The recombinant DNA technology allows the ectopic expression of nucleicacid sequences of interest, preferably gene sequences coding for aprotein product, in host cell systems which normally do not express saidgene sequences. This allows obtaining high levels of the protein ofinterest for the therapeutic, industrial or research use.

The ectopic expression, or else the forced expression of one or morenucleic acid sequences of interest in cell systems which normally do notexpress said nucleic acid sequences, is done by inserting at least onenucleic acid sequence of interest, for example at least one genesequence, in an expression vector which then will be introduced in theselected host cell system.

The known host cell systems are both of prokaryotic origin, usuallybacteria, and of eukaryotic origin, more complex both animal and plantcell systems, and the selection of the cell system depends on the typeof protein which is intended to be expressed. The ectopic expression inthe bacteria is the best option for producing high quantities ofproteins at low production costs. However, eukaryotic systems need to beused when the protein requires post-translational modifications to keepits biological activity.

The expression vector is a nucleic acid molecule able to transport anddrive the expression of DNA sequences of interest inside a host cellsystem.

It is imperative that the expression vector contains all the signalsneeded to the host cell system to transcribe the gene sequence ofinterest.

Among the signals the expression vector must contain, a key element isrepresented by the promoter.

The promoter is a nucleotide sequence which is upstream of the sequencecoding for the gene of interest and is recognized by the RNA polymeraseof the host cell system, which allows to start the transcription ofsequences placed immediately downstream of it.

From the functional point of view, the promoter performs the samefunction both in the eukaryotic genomes and in the prokaryotic(bacterial) genomes but, from the structural point of view and theorganization of the recognition sequences, the prokaryotic andeukaryotic promoters are definitely different.

At present the problem of the selection of the cell system requires thatmany different expression vectors must be used and “designed” ad hocbased on the cell system in which they will be used. To date, promotersequences are not known which allow obtaining a high expression level ofgenes of interest both in prokaryotic cell systems and in eukaryoticcell systems. In fact, promoter sequences able to allow a highexpression level of genes of interest in prokaryotic cell systems and inthe yeast, which is an eukaryotic cell system, are not known.

EP2772539 describes two nucleic acid sequences of promoters able toexpress genes of interest in prokaryotic and eukaryotic cell systems.

However, said promoters show limitations both in terms of efficiency andin terms of functionality for the expression of the gene of interest inthe yeast. Therefore, there's particular need of promoter sequences ableto activate the expression of the gene downstream of them both inprokaryotic cells and in eukaryotic cells (including yeast).Furthermore, there is the need of expression vectors allowing highexpression levels of genes in prokaryotic cell systems and in eukaryoticcell systems of different organisms.

OBJECTS OF THE INVENTION

It is a purpose of the present invention to provide a sequence havingtranscription promoter activity of a nucleic acid sequence of interest,for example a gene, in prokaryotic cell systems and eukaryotic cellsystems.

It is also a purpose of the present invention to provide an expressionvector which allows obtaining high expression levels of a nucleic acidsequence of interest in prokaryotic cell systems and eukaryotic cellsystems.

It is another purpose of the invention to provide a method forexpressing a sequence of interest in a prokaryotic and eukaryotic hostcell system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the transposable element P ofDrosophila melanogaster from which the sequence of the invention hasbeen derived.

FIG. 2 depicts the expression vector of the invention, comprising thesequence of the invention.

FIG. 3 depicts the results of a bioluminescence assay carried out infour cell systems by comparing different expression vectors with theexpression vector comprising the sequence of the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (191083US2.xml; Size:8,311 bytes; and Date of Creation: Dec. 22, 2022) is herein incorporatedby reference in its entirety.

DESCRIPTION OF THE INVENTION

The aforementioned purposes are achieved by the object of the presentinvention, i.e. a nucleic acid sequence having transcription promoteractivity in prokaryotic and/or eukaryotic cell systems.

The transcription is the transfer process of the genetic informationfrom the DNA to the RNA which then will be translated into the protein.

The nucleotide sequence of the invention, having transcription promoteractivity in prokaryotic and eukaryotic cell systems, has beenarbitrarily called “UnGE” (English acronym of “Universal GeneExpression”).

Thus, within the present description we can refer to the nucleotidesequence of the invention having transcription activator activity inprokaryotic and eukaryotic cell systems also with the acronym “UnGE” or“UnGE promoter sequence”.

The UnGE promoter sequence of the invention has sequence SEQ. ID. NO. 1:

-UnGE (SEQ. ID. NO. 1) CATGATGAAATAACATAAGGTGGTCCCGTCGATAGCCGAAGCTTACCGAAGTATACACTTAAATTCAGTGCACGTTTGCTTGTTGAGAGGAAAGGTTGTGTGCGGACGAATTTTTTTTTGAAAACATT AACCCTTACGTGGAATAAAAAAAA

The sequence according to the invention comprises at least one 5′ITRnucleotide sequence of the transposase gene of Drosophila melanogaster.

In the present invention, by the term “5′ITR” is meant to denoteinverted terminal repeats in 5′ of the transposase gene comprised in a Pelement of Drosophila melanogaster.

In molecular biology, a P element is a type of transposable elementpresent in the genome of the Diptera Drosophila melanogaster.

The transposable element, also called transposon, is a nucleic acidsequence present in the prokaryotic and eukaryotic genomes able to movefrom one position to another one of the genome.

The P element comprising the sequence of the invention is a class IItransposon. The canonical length of said P elements is 2907 bp and theyinclude the gene coding for the transposase, which is an enzymecatalyzing the excision and integration reactions of the transposableelement. Therefore, said P elements are autonomous transposons, able tocarry out the transposition thanks to the transposase they code for.

The P element comprises inverted terminal repeats (ITR) flanking thetransposase gene. Said inverted terminal repeats (ITR) are sequences of31 bp in 5′ and 3′ of the transposase gene (FIG. 1 ). Therefore, 5′ITRsequences are inverted terminal repeats in 5′ of the transposase gene ina P element of Drosophila melanogaster.

The UnGE promoter sequence according to the invention comprises at leastone 5′ITR of a P element of Drosophila melanogaster, the length of said5′ITR being 31 bp.

By the term “5′ITR nucleotide sequence derived from the P element ofDrosophila melanogaster” is meant herein to refer to all the nucleicacid sequences having a similarity percentage equal to at least 70%,preferably 80%, even more preferably 95% with that of the Drosophilaspecies belonging to the Drosophila genus.

By way of example, the 5′ITR nucleic acid sequences derived from the Pelement of other Drosophilidae with a similarity percentage equal to atleast 70%, preferably 80%, even more preferably 95% with the 5′ITRnucleotide sequence derived from the P element of Drosophilamelanogaster are considered sequences similar to the 5′ITR nucleotidesequence derived from the P element of Drosophila melanogaster.

Advantageously, it has been found that the UnGE promoter sequence of theinvention includes an activating sequence of the transcription of thegene coding for the transposase of the P element.

The length of the UnGE promoter sequence of the invention is between 100bp and 200 bp, preferably between 120 and 160 bp, even more preferablythe length is 152 bp.

Advantageously, the use of an UnGE promoter sequence whose the length isbetween 100 bp and 200 bp, preferably between 120 and 160 bp, even morepreferably is 152 bp, allows obtaining good expression level of nucleicacid sequences of interest in prokaryotic and/or eukaryotic cellsystems. Therefore, said UnGE promoter sequence can be used for theectopic expression of nucleic acid segments of interest in prokaryoticand eukaryotic cell systems.

According to the invention, the UnGE promoter sequence is between thefirst nucleotide and the nucleotide preceding the first ATG codon of thetransposase gene inside the P element of Drosophila melanogaster. TheATG codon (triplet AUG on the mRNA) is the specific sequence having 3nucleotides (triplet) which is used as starting codon of the translationof the mRNA into protein. ATG codon codes for methionine.

Therefore, methionine is the amino acid which occupies the N-terminal ofall the proteins of the eukaryotes and archaeobacteria.

According to the invention, the UnGE promoter sequence can be used forthe activation of the transcription of at least one nucleic acidsequence of interest in four different host cell systems, for example inprokaryotic host cells and/or eukaryotic host cells. By “host cellsystem” is meant herein to denote the cell in which nucleic acidsequences of interest, for example gene sequences, are introduced byexpression vectors.

Surprisingly, in fact it has been found that the UnGE promoter sequenceof the invention is able to operate at the same time as promoter in fourdifferent cell systems. In particular, the UnGE promoter sequence isable to activate the transcription of the luciferase gene in bacteria,mammalian cells, yeast and insect cells (Example 2). The luciferase isan enzyme coded by the luc gene of Photinus pyralis, used as reporterfor the quantitative analysis of elements potentially regulating thegene expression, including the strength of a promoter. The luciferase isalso used as reporter protein for the study of the interaction amongproteins and nucleic acids and the protein-protein interactions.Therefore, according to the invention, the UnGE promoter sequence isused for the activation of the transcription of at least one nucleicacid sequence of interest in prokaryotic cells, such as for examplebacteria, and in eukaryotic cells, such as for example mammalian cells,yeast and insect cells.

According to the invention, the UnGE promoter sequence of the inventionis used for the activation of the transcription of at least one nucleicacid sequence of interest when inserted in an expression vector, saidexpression vector being then introduced in host cell systems. Accordingto an embodiment, the UnGE promoter sequence of the invention is usedfor the expression of nucleic acid sequences of interest non-coding forproteins. In fact, it is known that some non-coding genome regions, areresponsible for the production of regulatory RNAs of the geneexpression, such as for example the miRNAs. Said miRNAs are endogenousmolecules of non-coding RNAs active in the regulation of the geneexpression at the transcriptional and post-transcriptional level.

According to the invention, the UnGE promoter sequence is placedupstream of nucleic acid sequences of interest.

Therefore, in an embodiment, the UnGE promoter sequence of the inventioncan be placed upstream of nucleic acid non-coding sequences, in order tostudy the function of new elements of the genome in the basic research.

According to a particularly preferred embodiment, the UnGE promotersequence of the invention is used for the activation of thetranscription of at least one gene sequence of interest. According tosaid particularly preferred embodiment, the UnGE promoter sequence ofthe invention is placed upstream of said gene sequences of interest. Inparticular, said gene sequences of interest are sequences coding for aprotein product of interest and comprise the exons of said gene.

The exon is the gene (eukaryotic or of archaeobacteria) part which istranscripted in RNA, together with the introns. Subsequently, by aprocess defined splicing, the introns are removed, whereas the exons arelinked in the mature RNAs and translated into an amino acid sequence.

It has been observed that the UnGE promoter sequence of the inventionhas a weak promoter activity. A weak promoter is a promoter able tomoderately activate the expression of the sequence of interest placeddownstream of it, such as for example a gene. Therefore, said UnGEpromoter sequence of the invention is particularly suitable to be usedfor the expression of proteins of which a medium-low level is desired.Advantageously, the UnGE promoter sequence of the invention isparticularly suitable to be used for the expression of proteins which,in high amounts, are toxic for the cell.

Therefore, the UnGE promoter sequence of the invention can be used asweak promoter for the expression of coding sequences or non-codingsequences in prokaryotic and eukaryotic cell systems. For example, theUnGE promoter sequence can be used as weak promoter in prokaryotic cellsystems, such as bacteria, and in eukaryotic cell systems, such asmammalian cells and yeast.

Advantageously, it has been observed that the UnGE promoter sequence ofthe invention doesn't have weak promoter activity when it is used aspromoter for the expression of sequences of interest in cells ofinterest.

Therefore, advantageously, the UnGE promoter sequence can be used ininsect cell systems for the expression of proteins of which a high levelis desired.

The UnGE promoter sequence of the invention is suitable to be used forthe preparation of expression vectors.

It is also an object of the present invention an expression vectorcomprising the UnGE promoter sequence of the invention. An expressionvector according to the invention is a nucleic acid molecule able tocarry a nucleic acid sequence in a prokaryotic and/or eukaryotic hostcell and able to obtain the expression of said nucleic acid sequence insaid host cells. The expression vector comprising the UnGE promotersequence of the invention has been arbitrarily called “pUnGE”. Thus,within the present description we can refer to the expression vectorcomprising the UnGE promoter sequence also with the acronym “pUnGE”.

According to the invention, the expression vector pUnGE comprises theUnGE promoter sequence (SEQ. ID. NO. 1) upstream of a nucleic acidsequence of interest. According to the invention, the expression vectorpUnGE further comprises a selection marker, such as for example anantibiotic resistance gene, and an origin of bacterial replication.

According to a particularly preferred embodiment, the expression vectorpUnGE comprises the UnGE promoter sequence upstream of a nucleic acidsequence of interest, said nucleic acid sequence of interest being agene of interest (transgene). By the term expression vector is meantherein to refer to any nucleic acid molecule for cloning andtransferring a nucleic acid in a host cell. By the term “cloning” ismeant herein to denote inserting DNA sequences into expression vectorsable to transcribe the sequence/gene of interest inside the host cell inwhich they are inserted. For example, expression vectors according tothe invention are considered the plasmid vectors, cosmids, phages,bacterial artificial chromosomes (BAC), yeast artificial chromosomes(YAC), and viral vectors. In FIG. 2 a generic expression vectoraccording to the invention is depicted.

The expression vectors according to the present invention can beprepared according to methods known in the art.

The expression vectors pUnGE according to the present invention overcomethe limits of the known art as they allow the transcription of nucleicacid sequences of interest, for example genes, both in prokaryotic cellsand eukaryotic cells, without the need to use genetically engineeredhost systems.

As it is possible to observe in FIG. 3 , the expression vectorcomprising the promoter sequence according to the invention (depicted inFIG. 3 as UnGEp) is able to express the nucleic acid sequence ofinterest, in the case in question the luciferase gene, also in yeastcells, advantageously at a greater extent with respect to the vectorcomprising the promoter of the known art UniBa1 upstream of the gene forthe luciferase (denoted in FIG. 3 as Ba1). Furthermore, the expressionvector of the invention is able to express the luciferase gene moreefficiently than the vectors comprising the promoters of the known artUniBa1 and UniBa3 also in mammalian cells, in insect cells and inbacteria.

In light of this, it follows that the expression vectors according tothe invention can easily be used for the transfection techniques of anycell line, for example for cultured cell lines for basic researchstudies on the gene expression, for the production of recombinantproteins or toxicological and pharmacological studies.

By “transfection” is meant herein to denote the introduction process ofexogenous biological material in eukaryotic cells, in most casesmammalian cells. The transfection process can be carried out in vitro ontarget cells in long-term cell cultures, ex vivo on cells isolated froman organism and transferred on culture medium and in vivo directly oncells of an organism.

Furthermore, the expression vectors according to the invention caneasily be used for the bacterial transformation and bacterialtransduction techniques.

By “transformation” is meant herein to denote a molecular biologytechnique used to introduce genetic material in bacteria cells.

By “bacterial transduction” is meant to denote the passage of the DNA ofa bacterium to another by a phage. A phage, also called bacteriophage,is a virus able to infect the bacterial cells. This allows inserting, inthe bacterial genome, a sequence of interest present in the phage, forexample a gene sequence.

According to an embodiment, the expression vectors comprising the UnGEpromoter sequence of the invention are used for the gene therapy.

By “gene therapy” is meant herein to denote inserting, in specific hostcells, expression vectors comprising specific gene sequences of interestin order to cure diseases, preferably genetic diseases.

Therefore, the expression vectors based on the UnGE promoter for the usein gene therapy comprise at least one gene sequence of interestdownstream of the UnGE promoter sequence.

The UnGE promoter sequence has non-viral origin and, therefore, involvesreduced immunogenicity and cytotoxicity. This allows obtaining safeexpression vectors which are suitable for the use in gene therapy.

In another embodiment, the expression vectors of the invention can beused for the trans-kingdom gene therapy. The trans-kingdom gene therapyallows transferring the therapeutic material, in the form of nucleicacids and proteins, to mammalian cells by using cells belonging todifferent Kingdoms (for example bacteria and fungi) and by the use ofexpression vectors for the production of the therapeutic materialdirected to the target cells.

Therefore, by “therapeutic material” is meant herein to denote nucleicacids of interest and proteins of interest introduced in mammalian cellsin order to cure diseases.

Surprisingly, the expression vector pUnGE comprising the UnGE promotersequence is able to transcribe the DNA sequence of interest in bacterialcells in addition to eukaryotic cells, such as for example human, insectand yeast cells (Example 2).

It is a further object of the invention a method for expressing at leastone nucleic acid sequence of interest in a prokaryotic and/or eukaryotichost cell, which comprises the steps of:

-   -   a) cloning said at least one nucleic acid sequence of interest        in an expression vector comprising the UnGE promoter sequence        SEQ. ID. NO. 1;    -   b) introducing said expression vector in the host cell;    -   c) culturing said prokaryotic and/or eukaryotic host cell under        suitable conditions to obtain the expression of said nucleic        acid sequence of interest.

According to a preferred embodiment, the expression vector comprises theUnGE promoter sequence of the invention upstream of a gene sequence,transgene.

According to the invention, cloning the DNA sequences of interest insidean expression vector in step a) is carried out by known techniques.

According to the invention, step b) of introducing the expression vectorin the host cell can be carried out by transformation or transfection.

Step c) of cultivating the host cell under suitable conditions dependson the type of cell, e.g. prokaryotic cell or eukaryotic cell, andspecific cell sub-type, e.g. kidney cell and neuron. The cultureconditions of different specific cell sub-types are known in the artand, therefore, step c) of the method according to the invention can becarried out by using known culture conditions.

The use of the UnGE promoter sequence derived from the P element ofDrosophila melanogaster upstream of a sequence of interest produces anumber of unexpected advantages as it allows the activation of thetranscription of nucleic acid sequences of interest both in prokaryotichost cells and eukaryotic host cells by simplifying and optimizing theectopic expression procedures in different cell systems. Advantageously,the expression vector comprising the UnGE promoter sequence of theinvention is particularly suitable to be used for the expression ofproteins of which a medium-low level is desired. In particular, saidexpression vector is suitable for the expression of proteins which, inhigh quantities, are toxic for the cell.

Furthermore, said expression vector can be used for the study ofmodifiers of the gene expression acting directly on the promoters, suchas for example of the repressors or activators of transcription. Infact, by using the expression vector pUnGE comprising an UnGE promotersequence having weak promoter activity, the effect of the expressionmodifiers, for example having repression effects of the transcription,would immediately be evident by the drastic drop of the protein level.By using the expression vector pUnGE comprising an UnGE promotersequence having weak promoter activity, the effect of the expressionmodifiers, for example having activation effects of the transcription,would immediately be evident by the very large amount of protein.

Still more advantageously, the expression vector comprising the UnGEpromoter sequence of the invention is a valid alternative to theexpression vectors currently available. In fact, the sequence of theinvention and the expression vectors comprising it allow to quicklyswitch from experiments and assays in one cell system to another ofdifferent origin. It follows that the chosen procedures of the finalplatform for the expression of a sequence of interest, such as forexample a transgene (and related recombinant protein) will be greatlyaccelerated.

A further advantage arise from the fact that, by introducing theexpression vector based on the use of the UnGE promoter, the analysis ofthe gene expression or sequences of interest will be able to be carriedout in parallel after a single cloning operation in the pUnGE vector atthe level of prokaryotic cell systems and eukaryotic cell systems, forexample in mammalian cells, insect, yeast and bacterium cells. In otherwords, a unique vector comprising the nucleotide sequence of interestwill be able to be inserted in the four cell systems mentioned above tocarry out parallel experiments. Therefore, the UnGE promoter sequence ofthe invention and the expression vector comprising said sequence willallow reducing the research costs since it will no longer be necessaryto build or buy other expression vectors known for being used forspecific types of cells.

Advantageously, analysis and production times of recombinant products atany level (basic research, industrial production of therapeutic proteinsetc.) may also be reduced because only one expression vector comprisingan UnGE promoter sequence will be sufficient to carry out the analysisand production of proteins in multiple host cell systems.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a canonical P element ofDrosophila melanogaster. Said P element comprises the UnGE promotersequence of the invention in the element portion depicted by the bracethat includes the 5′ ITR portion. The transposase gene in a canonical Pelement comprises four exons (E0, E1, E2 and E3) and three intronsbetween E0 and E1, E1 and E2, and E2 and E3.

FIG. 2 depicts a generic plasmid expression vector comprising the UnGEpromoter sequence of the invention. In FIG. 2 are also schematized anorigin of bacterial replication (depicted in Figure as pUC ori) and anantibiotic resistance gene (depicted in Figure as Resistance Gene).

FIG. 3 depicts the results of a bioluminescence assay carried out withfive different expression vectors in four host cell systems. The hostcell systems are depicted on the abscissa of the graph and are humanHeLa cells, cells of Drosophila melanogaster S2R+, bacterium DH5α andyeast (BMA64).

The expression vectors used for the bioluminescence assay are alsodepicted on the abscissa of the graph. The expression vectors containingdifferent promoters upstream of the luciferase gene are depicted in FIG.3 as UnGEp (vector comprising the UnGE promoter sequence of theinvention), Ba1 (comprising the UniBal promoter sequence), Ba3(comprising the UniBa3 promoter sequence). Ba1 and Ba3 are expressionvectors known in the art to have promoter activity both in prokaryoticcell systems and eukaryotic cell systems. The activity of the UnGEpromoter sequence is compared with that of said promoters known in theart.

The negative control is depicted in FIG. 3 as “pGL3B”.

The positive controls are different for each host cell. In FIG. 3 , thepositive controls are depicted as SV40, copy, CAT and URA 3.

The bioluminescence results are expressed as relative light units %(Relative Light Units, RLU). The RLU % is depicted in the ordinate ofthe graph in FIG. 3 .

EXPERIMENTAL SECTION Example 1: Construction of the Expression VectorComprising the UnGE Promoter Sequence of the Invention

The UnGE promoter sequence of the invention (SEQ ID. NO. 1) has beencloned in the pGL3B-Basic plasmid expression vector (PromegaCorporation) upstream of the gene sequence of luciferase. SuchpGL3-Basic vector contains a reporter gene coding the luciferase enzymeof the Photinus pyralis firefly.

The plasmid has been cut with restriction enzymes XhoI and NcoI so as tobe linearized.

For the following experiment, the UnGE promoter sequence of theinvention has been amplified by primers having at their ends thesequences recognized by the restriction enzymes XhoI and NcoI.

Amplified UnGE promoter sequence obtained by using as template the“pCasper3” clone (Acc. No. GenBank U59055) and using the primers:

PrimerUnGE_fw (forward): (SEQ. ID. NO. 2)gatcctcgagCATGATGAAATAACATAAGGTG PrimerUnGE_rev (reverse):(SEQ. ID NO. 3) gatcccatggTTTTTTTTATTCCACGTAAGGGT

The PCR reactions to obtain the amplified UnGE promoter sequence havebeen carried out in standard conditions as mentioned.

Thermal Cycling Parameters

1 cycle:

Denaturation for 3′ at 94° C.

35 cycles:

Denaturation for 30″ at 94° C. Annealing for 30″ at 48° C. Elongationfor 20″ at 72° C.

1 cycle:

7′ at 72° C. 4° C.∞

The reaction mixture for the amplification of the UnGE promoter sequenceis constituted as follows:

Reaction buffer 1× Mg2+ 1.5 mM dNTP mix 0.2 mM primer UnGE_fw 0.4 μMprimer UnGE_rev 0.4 μM Platinum Taq 1 U Template DNA 1-5 ng Water up to50 μl

The enzyme used is Platinum Taq polymerase (Invitrogen, LifeTechnologies).

The oligonucleotides PrimerUnGE fw (forward) (SEQ. ID. NO. 2) andPrimerUnGE rev (reverse) (SEQ. ID. NO. 3) contain target sequences forthe restriction enzyme XhoI and NcoI (depicted in lowercase in theprimer sequences). The obtained amplification fragments have been clonedin the XhoI and NcoI sites of the plasmid vector pGL3-Basic (PROMEGA).

The enzymatic digestion of the amplified UnGE promoter sequence andpGL3-B vector has been carried out with the following method.

Digestion Reaction for the PCR Samples

Amplified UnGE 1 μg Buffer H (NEB) 1× Enzyme NcoI (NEB) 2 U Enzyme XhoI(NEB) 2 U Water up to 25 μl

The reactions have been carried out for 2 h at 37° C.

Digestion Reaction for the pGL3-B Plasmid

DNA pGL3-B 1 μg Buffer H NEB 10× 1× Enzyme NcoI NEB 2 U Enzyme XhoI NEB2 U Water up to 25 μl

The reactions have been carried out for 2 h at 37° C.

Ligation of the Digested Fragments

T4 DNA Ligase Reaction Buffer 1× Digested pGL3B 50 ng UnGE promotersequence 100 ng T4 DNA ligase (NEB) 1 U Water up to 20 μl

Reactions carried out at 15° C. for about 16 hours.

The recombinant plasmid clones have been purified with commercial kits(QIAGEN plasmid mini kit), quantified by measurement at Nanodrop andsubsequently 1 μg of each single plasmid has been carried to the cellsof interest.

The cloned fragment, i.e. the UnGE promoter sequence of the inventionand the luciferase gene downstream of said sequence, has been sequencedto verify the correctness of the sequence itself.

Example 2: Luciferase Assay for the Definition of the Activity of thePromoter of the Invention

An expression vector comprising the UnGE promoter sequence of theinvention, as it was generated in the example 1, has been used in theluciferase assay in four different cell systems. Said expression vectorcomprises the UnGE promoter sequence of the invention upstream of theluciferase gene and is called pUnGE.

To test the efficiency of the UnGE promoter sequence in thetranscription of the luciferase gene, a bioluminescence assay has beenfine-tuned.

In the bioluminescence assay, in each host cell system have been testedfive different expression vectors: three expression vectors eachcomprising a different promoter upstream of the luciferase gene, anegative control and a positive control. For all of the expressionvectors the commercially available plasmid named pGL3-Basic (PromegaCorporation) has been used. In fact, the pGL3-Basic plasmid isparticularly useful with the purpose of measuring the promoter activitythanks to the presence of a synthetic polyadenylation sequence upstreamof the multiple cloning site (MCS), reducing the transcriptionalbackground due to non-specific sequence.

The three expression vectors comprising three different promotersupstream of the gene for the luciferase are:

-   -   pGL3B-UnGE comprising the UnGE promoter sequence    -   pGL3B-Unibal comprising the UniBa1 promoter sequence, depicted        herein below.

UniBa1 (SEQ. ID NO. 4) CAGTCATGGTCAAAATTATTTTCACAAAGTGCATTTTTGTGCATGGGTCACAAACAGTTGCTTGTGCAGCAAGTGGGGGGAGGTGAAATGCAAAAAAACTTTTGCTTTTGCAAATTCAAACCTATGCAGAGTCAGATGAAAGAAGAATTGAAAAAATAACTGTTCCTATGCGCAAGGAAGAGGCAAATGAAGAGATCTTTATCAGTTGTCAGAAGTATTTGCACACGGTTTCGTCGCATCACAATTATTTTCACAACGCAATTTCTTCTTCAGTGATTGGTTTAGAGTGACAAGTGCCGGTTTGTTTGCTTAAATACATTTAAATTATTGAATAAAAATTAGATTTAATCATTTTCCTATTACAGTTATTAAATAAA

-   -   pGL3B-Uniba3 comprising the UniBa3 promoter sequence, depicted        herein below.

UniBa 3: (SEQ. ID. NO. 5) CAGAGGTGGTCAAAAGTAATTACACACCGAGCTTTTTTCTGCATGCTCAGCTTTAAAAGCTTGTTGTTGTTGTTGTTGTTCGCTGTTTTTGAAAAAAAGCTTTTGTGTCATAAAGCTTATTCTGTGTAGAAGCGATCGGCAAAAAGAAATTGCTAAAATAACGGTACAGGTTCGTAGATATAGCAAAAAGAACAGTTACCTAGAGGTTGTCAAAAGTATTTACACACATTTTTTTTGCGTCATAAGTATTTACACAATGTAAATTTGCCTTTAGTCTTTGATTTGCGTTGATCATGTGCGGTCTTTGACTAAGATCGGAATTCAATTTGTAGTTGAAATTGTAGA TGTGCTGTAAA.

UniBa1 and UniBa3 are two promoter sequences having activator activityof the gene transcription in prokaryotic and eukaryotic cell systems.Said sequences have been used as comparative sequences with which tocompare the activity of the UnGE promoter sequence.

The negative control is the plasmid not comprising a promoter upstreamof the luciferase gene (pGL3B).

The positive controls provide a bioluminescence reference value in thefour cell systems and are different for each host cell.

-   -   pGL3B-copy provides a bioluminescence reference value in insect        cell systems. It is a plasmid comprising the promoter of the        copy retro-transposon, widely used for the expression in        Drosophila. It has been amplified starting from the pCoBlast        plasmid, sold by Life Technologies as part of the DES®-Inducible        Kit expression system.    -   pGL3B-cat comprises the promoter of the CAT (chloramphenicol        acetyl transferase) gene and is used because it provides a        bioluminescence reference value in the bacteria. Said CAT        promoter has been amplified starting from the pHSG396 plasmid        containing the entire expression cassette for the        chloramphenicol acetyl transferase.    -   pGL3-promoter vector contains the SV40 promoter of viral origin        and represents the bioluminescence reference value in the human        HeLa cells. The pGL3-promoter vector plasmid is sold by Promega        (catalog number E1761).    -   pFL39-URA contains the promoter gel gene URA3 of yeast and        provides a bioluminescence reference value in the yeast. Said        URA promoter has been amplified starting from the pFL44 plasmid        containing the entire expression cassette of the URA3 gene of        Saccharomyces cerevisiae.

The plasmid DNAs have been extracted with commercial purification kits,quantified by reading on Nanodrop and, subsequently, 1 μg of each singleplasmid has been introduced in the host cell system of interest. Thetransfection method is different for eukaryotic and prokaryotic cells.For the transfection in eukaryotic cells, the MIRUS Translt_LT1 (MirusBio LLC) transfection agent has been used as specified in the suppliermanual, whereas in the case of prokaryotic cells, the procedure ofcalcium chloride has been used. Said expression vectors have beenintroduced in four cell systems: human HeLa cells, cells of Drosophilamelanogaster S2R+, E. coli DH5α bacterium and in S. cerevisiae BMA64yeast.

The cell systems used for the purpose of determining the activity of thepromoters in the tested expression vectors are:

-   -   DH5α™ (InvITRogen) (F-Φ801acZΔM15 A(lacZYA-argF) U169 recA1        endA1 hsdR17 (Rk−, Mk+) phoA supE44 λ-thi-1 gyrA96 relA1)    -   Drosophila S2R+(DGRC) derived from the Schneider's line 2        (Schneider, I. (1972). Cell lines derived from late embryonic        stages of Drosophila melanogaster. J. Embryol. exp. Morphol.        27:353-365)    -   HeLa cervical cancer cells (ATCC)    -   S. cerevisiae BMA64-1A (MAT a ura 3-1 ade 2-1 leu 2-3, 112        his3-11,15 trp1D can 1-100).

Each expression assay has been carried out in triplicate on a suitablenumber of cells, variable depending to the cell type used asexperimental model, but which always refers to procedures normally usedin analogous assays.

The expression level of the luciferase gene, and thus the amount of theprotein, has been evaluated based on the amount of light emitted. Infact, the luciferase is able to catalyze a particular chemical reaction,during which chemical energy is converted into light energy.

The bioluminescence results are expresses as relative light units %(Relative Light Units, RLU in the ordinate).

RLU % detected is directly proportional to the level of synthesizedprotein and, therefore, to the expression level of the luciferase genedownstream of the various tested promoters. RLU % of the positivecontrol was arbitrarily set equal to 100%.

As it is possible to observe in FIG. 3 , the expression vector pUnGEcomprising the UnGE promoter sequence (depicted in FIG. 3 as UnGEp) inDrosophila cells gives a high expression level of the luciferase gene,higher than that of the positive control. In fact, the % of relativelight units detected is equal to at least 450%.

Furthermore, from FIG. 3 it is possible to observe that the UnGEppromoter is able to express the sequence of interest, luciferase gene,also in yeast cells, advantageously at a greater extent than the vectorcomprising the UniBa1 promoter upstream of the gene for the luciferase(depicted in FIG. 3 as Ba1). Furthermore, the pUnGE expression vectorcomprising the UnGE promoter sequence of the invention is able toexpress the luciferase gene more efficiently than the UniBa1 and UniBa3promoters known in the art (contained in the pUniBa1 and pUniBa3expression vectors) also in mammalian cells, insect cells and bacteria.

1. Method of performing gene therapy with the expression vectorcomprising an isolated nucleic acid sequence having SEQ. ID. NO.1, saidmethod comprising the step of inserting, in specific host cells, saidexpression vector in order to cure genetic diseases.
 2. The methodaccording to claim 1 wherein said method is trans-kingdom gene therapy.3. A method for expressing at least one nucleic acid sequence ofinterest in a prokaryotic and/or eukaryotic host cell, comprising thesteps of: a) cloning said at least one nucleic acid sequence of interestin an expression vector according to claim 2; b) introducing saidexpression vector in the host cell; c) culturing said prokaryotic and/oreukaryotic host cell under suitable conditions to obtain the expressionof said nucleic acid sequence of interest.
 4. The method according toclaim 1, wherein the isolated sequence has the function of transcriptionpromoter of at least one nucleic acid sequence in prokaryotic and/oreukaryotic cell systems.
 5. The method according to claim 1, wherein theisolated sequence comprises at least one 5′ITR of a P element ofDrosophila.
 6. The method according to claim 5, wherein the isolatedsequence comprises at least one 5′ITR of a P element of Drosophilamelanogaster.
 7. The method according to claim 5, wherein the length ofsaid at least one 5′ ITR of a P element of Drosophila is 31 bp.
 8. Themethod according to claim 1, wherein the isolated sequence has a lengthof between 100 bp and 200 bp.
 9. The method according to claim 1,wherein the isolated sequence has a length of between 120 and 160 bp.10. The method according to claim 1, wherein the isolated sequence has alength of 152 bp.
 11. The method according to claim 3, wherein saidprokaryotic systems are bacteria and said eukaryotic systems areselected from mammalian cells, yeast and insect cells.